Related National Programs

1a. Objectives (from AD-416)
We lack a clear understanding of C and N flux in switchgrass plants and other perennial C4 grasses over a growing season and the underlying controlling gene expression patterns in the crown, rhizomes, and dormant buds which are the critical tissues required for generating above-ground tillers every growing season. Processes controlling the cycling of C and N into crown and root tissue at the end of the growing season are also not known. Developing a detailed understanding of the seasonal changes of gene expression coincident with major fluxes of C and N into and out of the crown and rhizomes will yield important insights into these processes. We will use 454 sequencing technology to profile barcoded cDNA libraries derived from crowns, rhizomes and dormant buds during critical phases of transition and will employ stable 13C and 15N isotope enrichment for C and N utilization, sequestration, and cycling analysis. Additionally, we will perform metabolic profiling of sugars, starch, phenolics, protein, amino acids and lipids.
Genotypes and cultivars with known differences in winter survival and pre-frost maturity or senescence differences will be utilized to identify key genetic mechanisms involved in controlling these events. Complete winter survival followed by rapid spring growth to optimize light or energy capture are two key breeding goals for perennial herbaceous energy crops. Identification of genetic, physiological and biochemical control over these processes will accelerate our ability to breed new cultivars as well as increase our understanding of many aspects of the basic biology of the switchgrass plant. We have developed a high yielding lowland switchgrass with significantly improved winter survival in comparison to its parent population. We will utilize these populations to develop a greater understanding of C and N cycling as it affects winter survival. In addition, we have developed unique Upland switchgrass populations by recurrent breeding for forage digestibility which is strongly associated with low lignin concentration in which winter survival and plant fitness has been significantly decreased. The same 454 analysis platform as described above will be used on populations and genotypes with known differences in winter survival.
Ultimately we would like to uncover marker-trait associations that can be used to reduce the generations, and within generation time and expense of phenotyping in the breeding process by use of marker assisted selection. The transcript profiling that we will undertake will provide access to a number of candidate genes. Additional evidence of these genes involvement will be required and will be obtained through an association study controlling for false positives arising from population structure through the use of previously mapped EST-SSR markers.

1b. Approach (from AD-416)
C and N Partitioning and recycling in switchgrass over two growing seasons using stable isotope methods, transcript profiling and metabolomics of crown and rhizome tissues:
Plants from two contrasting tetraploid backgrounds: cv Kanlow (Lowland, high yield, not well adapted to Upper Midwest); and cv Summer, (Upland, lower yield and in a different heterotic group than Kanlow) and Kanlow N1, which is a Lowland cold-adapted, high yield population selected from a Kanlow base population with significantly improved winter survival, will be planted in a field nursery as well as in large pots maintained under ambient atmospheric conditions. For C-cycling, replicated rows of plants in the field will by pulsed labeled with 13CO2 during the growing season (mid vegetative approximately mid/late May, three labeling/week for 1 week to obtain well labeled plants; a small subsample will be harvested 2 days and 7 days post-labeling to confirm isotope enrichment). Above ground plant parts will be harvested at specific times (see below) after labeling, separated into leaves, sheaths and stems, dried, ground and analyzed for 13C- enrichment using stable-isotope ratio mass spectrometry. For N-recycling, 15N will be supplied as ammonium nitrate just prior to green-up (mid April) and above ground plant materials will be harvested, separated and analyzed as described above.
Gene Profiling During Regreening and Dormancy of Bulked Segregants:
The populations selected for this objective have been growing in replicated field nurseries in NE, and select genotypes with low winter survival have been clonally maintained in greenhouses and represent a critical and unique resource for discoveries that affect plant fitness. Initially we will bulk 10 plants each with the most divergent scores for winter survival and spring regrowth RNA isolated from the crown, rhizomes, and dormant buds from three harvest dates – early spring at the first sign of regreening, post anthesis, and following first killing frost will be pooled and bulked by genotype. These RNAs will be analyzed as above by the creation of barcoded cDNA libraries and 454.
Creation and Analysis of QTL and Association Mapping Panel in Breeding Stock Developed for the Northern Plains Climate that Show Significant Heterosis:
There are 148 single-seed descent tetraploid plants in a marker population derived from a cross between an upland cv Summer and an adapted lowland cv Kanlow plant. This population will be expanded to 250 individuals and replicated in an alpha lattice design, along with 500 tetraploid upland and lowland lines derived from a foundation nursery that has been randomly intermated for two generations and high yielding stock lacking cold tolerance. Material from this association panel will be evaluated for biomass yield, rapid spring regrowth, and heading date. Using available mapped EST-microsatellites will enable evaluation of population structure, and based on sequence profiling, we will resequence up to 10 kb from selected candidate loci in the 500 individual association panel.

3. Progress Report
Significant parts of the research are being conducted by University of Nebraska cooperating scientists who are being funded via a subsidiary grant (Project 5440-2100-028-02G).
This work is being performed through a Department of Energy competitive grant awarded in May 2009 with an official start date of January 1, 2010 through December 31, 2012. For FY 11 both laboratory and field experiments were conducted. All team members were cognizant of the planned experiments and had approved implementation. Communication was by email and telephone between the ADODR and appropriate project personnel at distant locations (ARS-Albany and University of Nebraska-Kearney) and by in-person meetings for project personnel located in Lincoln, NE.
Plant materials labeled with C13 CO2 in spring FY10 were collected throughout the growing season, separated into respective plant parts, oven dried and ground. Several samples were analyzed by mass spectrometry to gauge the level of incorporation of C13 into plant parts. Initial analysis showed that plants were labeled adequately and there was an expected dilution of label during the period of active growth. Crown + rhizomes were harvested from 4 populations at selected times over the growing season, cleaned and flash frozen in liquid nitrogen. Roots were also collected from each sample at each harvest date. All frozen plant materials were stored at -80C for future RNA analyses by high-throughput sequencing. High-throughput sequencing of previously collected crown materials from cv Summer and cv Kanlow was performed using two platforms, 454 FLX and Illumina sequencing by synthesis strategies. The ~ 1 million sequences obtained from cv Summer crowns + rhizomes using the 454 platform was used to assemble a preliminary crown + rhizome transcriptome. Analysis of these sequences is largely complete, and a manuscript is being prepared for submission to a peer-reviewed journal in FY11.
In May 2011, plants were labeled with N15-enriched urea just prior to green-up. Some of these plants were also labeled with 13CO2 in June 2011. Plant materials will be collected throughout the FY11-FY12 growing season, dried and analyzed for N15 and C13.